Preparation of 125-I-labeled human thyroxine-binding alpha globulin and its turnover in normal and hypothyroid subjects.

A protein with the electrophoretic, immunologic, and hormone-binding properties of thyroxine-binding globulin (TBG) has been prepared from human plasma and labeled with radioiodine (125-I) by an enzymatic method of iodination. The [125-I]TBG retained the electrophoretic and immunologic characteristics of unlabeled TBG but exhibited a partial loss of thyroxine-binding activity, as assessed by affinity chromatography. The in vivo behavior of [125I]TBG was studied in six euthyroid subjects (controls) with normal serum levels of TBG as measured both by radioimmunoassay and by determination of maximal T4-binding capacity and in four male patients with untreated primary hyperthyroidism, three of whom had elevated serum TBG. The half-time of the final slope of the plasma disappearance curve averaged 5.0 days plus or minus 1.2 (SD) in the controls and ranged from 3.9 to 109 days in the hypothyroid patients. The distribution volume was similar in the two groups, 6.7 plus or minus 1.3 vs. 7.1 plus or minus 2.1 liters. The catabolic clearance rate averaged 0.99 plus or minus 0.33 liters plasma/24 h in the controls and 0.92 plus or minus 0.46 in the hypothyroids. The absolute turnover rate of TBG, calculated from the catabolic clearance rate multiplied by the serum concentration of radioimmunoassayable TBG, averaged 17.8 plus or minus 2.1 mg/day in the controls and ranged from 14.8 to 33.2 mg/day in the hypothyroids. Among the entire group of subjects there was no correlation between the serum TBG concentration and the absolute turnover rate of TBG.

The distribution kinetics of triiodothyronine: studies of euthyroid subjects with decreased plasma thyroxine-binding globulin and patients with Graves' disease.

The kinetics of distribution of 3,3',5-triiodo-L-thyronine (T(3)) have been studied employing both a single-injection and a continuous infusion of T(3-) (131)I. External monitoring of radioactivity in the liver during the infusion permitted estimation of the hepatic distribution volume (V(H)) and the one-way hepatic clearance (C(H)) of the hormone. Among 10 euthyroid control subjects, V(H) averaged 2.07 liters +/-0.50 (SD), and the mean value for C(H), 231 ml of plasma per min (+/-64). In three euthyroid men whose plasma showed decreased binding capacity by thyroxine-binding globulin (TBG) abnormally high V(H) and C(H) values were found, the increase in C(H) being proportional to the decrease in binding activity by plasma proteins. Among all 13 subjects, there was a high correlation (+ 0.86) between C(H) and the proportion of free hormone in plasma, measured in vitro. In four patients with hyperthyroid Graves' disease V(H) ranged from 3.2 to 4.2 liters and C(H) was elevated in every case, averaging 989 ml/min. The increase in C(H) in this group was out of proportion to the elevation of free hormone fraction in plasma. Seven patients who were either euthyroid or hypothyroid after treatment of Graves' disease showed a slight but significant increase in C(H) compared with the euthyroid controls without Graves' disease. The percentage of free hormone in the plasma of the treated group was normal or low and therefore could not explain the persistent elevation in unidirectional hepatic clearance observed. The rate of accumulation of labeled T(3) in the tissues of the thigh during the interval from 10 to 60 min of the sustaining infusion of tracer was slow compared to the rate of equilibration in the liver and did not differ significantly among the various groups studied. These latter findings suggest that in slowly equilibrating tissues such as the thigh the kinetics of T(3) distribution are relatively insensitive to alterations in hormone-binding activity by plasma proteins.

3,3'-Diiodothyronine production, a major pathway of peripheral iodothyronine metabolism in man.

3,3'-Diiodothyronine (3,3'-T(2)) has been detected in human serum and in thyroglobulin. However, no quantitative assessment of its clearance rate (CR), production rate (PR), or of the importance of extrathyroidal sources of 3,3'-T(2) relative to direct thyroidal secretion is yet available. This study examines these parameters in seven euthyroid subjects, and in eight athyreotic subjects (H) eumetabolic due to thyroxine therapy (HT(4)) (n = 5) or triiodothyronine replacement (HT(3)) (n = 3). A highly specific radioimmunoassay for the measurement of 3,3'-T(2) in whole serum was developed. Serum 3,3'-T(2) concentrations were (mean +/- SD) 6.0+/-1.0 ng/100 ml in 13 normal subjects, 9.0+/-4.6 ng/100 ml in 25 hyperthyroid patients, and 2.7+/-1.1 ng/100 ml in 17 hypothyroid patients. The values in each of the latter two groups were significantly different from normal. 3,3'-T(2) was detected regularly in normal concentrations in 11 hypothyroid patients eumetabolic by treatment with synthetic T(4), in 10 eumetabolic patients suffering from nonthyroidal systemic illness, and in 2 subjects with elevated serum T(4)-binding globulin. The 3,3'-T(2) CR was assessed from data acquired from the (125)I-3,3'-T(2) constant infusion technique. The 3,3'-T(2) PR was calculated from CR and serum concentration of 3,3'-T(2) determined by radio-immunoassay. In the HT(4) subjects the 3,3'-T(2) CR averaged 840+/-377 liters/day and 3,3'-T(2) PR 33.9+/-12.5 mug/day. These results were not significantly different from those in the control group: 3,3'-T(2) CR 628+/-218 liters/day and 3,3'-T(2) PR 39.8+/-19.8 mug/day (all corrected to 70 kg body wt). In addition to 3,3'-T(2) PR, T(3), and reverse triiodothyronine (rT(3)) PR were determined in three of the HT(4) subjects. In each case studied, the 3,3'-T(2) PR was close to the combined triiodothyronine (T(3) + rT(3)) PR. The mean molar ratio of T(2) PR/(T(3) + rT(3)) PR was 1.08+/-0.10. The results obtained in the HT(4) subjects indicate that the production of 3,3'-T(2) is a major route of T(4) metabolism. The combined studies of 3,3'-T(2), T(3) and rT(3) PR in the HT(4) subjects indicate that both T(3) and rT(3) are major precursors of 3,3'-T(2). In the HT(3) subjects, the conversion of T(3) to 3,3'-T(2), determined as the molar ratio of 3,3'-T(2) PR to T(3) PR, ranged from 0.36 to 0.92, providing further evidence that T(3) is a precursor of 3,3'-T(2). From the close agreement between the mean values for 3,3'-T(2) PR in the euthyroid and HT(4) group it is concluded that most, if not all of the 3,3'-T(2) produced in normal humans is derived by extrathyroidal conversion from T(3) and rT(3).

Thyroxine transport and distribution in Nagase analbuminemic rats.

The postulate that thyroxine (T4) in plasma enters tissues by protein-mediated transport or enhanced dissociation from plasma-binding proteins leads to the conclusion that almost all T4 uptake by tissues in the rat occurs via the pool of albumin-bound T4 (Pardridge, W. M., B. N. Premachandra, and G. Fierer. 1985. Am. J. Physiol. 248:G545-G550). To directly test this postulate, and to test more generally whether albumin might play a special role in T4 transport in the rat, we performed in vivo kinetics studies in six Nagase analbuminemic rats and in six control rats, all of whom had similar serum T4 concentrations and percent free T4 values. Evaluation of the plasma disappearance curves of simultaneously injected 125I-T4 and 131I-albumin indicated that the flux of T4 from the extracellular compartment into the rapidly exchangeable intracellular compartment was similar in the analbuminemic rats (51 +/- 21 ng/min, mean +/- SD) and in the control rats (54 +/- 15 ng/min), as was the size of the rapidly exchangeable intracellular pool of T4 (1.13 +/- 0.53 vs. 1.22 +/- 0.36 micrograms). This latter finding was confirmed by direct analysis of tissue samples (liver, kidney, and brain). We also performed in vitro kinetics studies using the isolated perfused rat liver. The single-pass fractional extraction by normal rat liver of T4 in pooled analbuminemic rat serum was indistinguishable from that of T4 in pooled control rat serum (10.9 +/- 3.3%, n = 3, vs. 11.4 +/- 3.4%). When greater than 98% of the albumin was removed from normal rat serum by chromatography with Affi-Gel blue, the single-pass fractional extraction of T4 (measured by a bolus injection method) did not change (16.3 +/- 2.1%, n = 5, vs. 15.2 +/- 2.5%). These data provide the first valid experimental test of the enhanced dissociation hypothesis and indicate that there is no special, substantive role for albumin in T4 transport in the rat.

Thyroxine (T4) transport and distribution in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from transthyretin.

To test whether plasma transthyretin (TTR) might play a specific direct role in the transfer of T4 from the plasma to tissues, in vivo kinetic studies were performed in control rats and in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from TTR. The plasma disappearance curves of simultaneously injected [125I]T4 and [131I]albumin were analyzed to determine the rate constant for the transfer of T4 from the extracellular compartment to the rapidly exchangeable intracellular compartment (KE) and the steady state distribution ratio of T4 between the rapidly exchangeable intracellular compartment and the extracellular compartment (Imax/Emin). When rats were injected ip with EMD 21388 (2 mumol/100 g BW), the free T4 fraction in serum increased approximately 8-fold. This was due to displacement of T4 from TTR, as assessed by electrophoresis of serum proteins in the presence of [125I]T4. Concomitantly, both KE and Imax/Emin increased 6-fold in the treated rats. These results fail to confirm a major specific role for TTR in the transfer of T4 from the plasma to tissues. Instead, they are consistent with both the free hormone transport hypothesis and the free hormone hypothesis in this setting.

Glucose and insulin reverse the effects of fasting on 3,5,3'-triiodothyronine neogenesis in primary cultures of rat hepatocytes.

The cellular mechanisms by which carbohydrate refeeding reverses the effect of fasting on T3 metabolism were studied in primary cultures of hepatocytes (24 h) harvested from 48-h fasted rats. Net T3 neogenesis (T3 generated from T4) in the fasted hepatocyte preparations (9.2 +/- 0.9 pmol/min X 100 mg protein) was significantly less (P less than 0.001) than that in hepatocyte cultures derived from 72-h glucose-fed rats (41 +/- 0.8 pmol/min X 100 mg protein). Preincubation (18 h) with either glucose (2.5-10 mM) or insulin (10-500 nM) significantly increased the fasted hepatocyte T3 levels to 28 +/- 0.6 and 22 +/- 1.3 pmol/min X 100 mg protein, respectively. Furthermore, incubation with both of these agents demonstrated a greater effect on hepatic T3 neogenesis than with either alone. Fasted hepatocyte T3 neogenesis was enhanced by enrichment with dithiothreitol (5 mM), but the T3 generation remained significantly less than that in cells exposed to glucose or insulin. Studies with glucose analogs demonstrated that preincubation with 2-deoxyglucose (5 mM) significantly increased (P less than 0.001) hepatocyte T3 neogenesis, but 3-O-methylglucose (5 mM) had no effect. In contrast, the insulin-mimetic compounds Concanavalin-A or spermine did not stimulate T3 neogenesis in the fasted hepatocyte cultures. Thus, rat hepatocytes sustained in primary culture for 24 h retain the T3 metabolic characteristics of the intact animal. Glucose and insulin reverse the effect of fasting on hepatocyte T3 neogenesis. The additive response to glucose and insulin suggests that T3 neogenesis is modulated through different mechanisms. The replication of the glucose effect by 2-deoxyglucose and the inability of dithiothreitol to reverse the effect of fasting on hepatocyte T4 5'-deiodinase activity suggest that neither intermediates in the glycolytic pathway nor thiol cofactors mediate the glucose effect. Thus, the restoration of liver T3 metabolism consequent to carbohydrate refeeding of the fasted rat may be mediated by the glucose and insulin responses.

Uptake of 3,5,3'-triiodothyronine by the perfused rat liver: return to the free hormone hypothesis.

To investigate the mechanism by which T3 in plasma enters hepatic cells, we measured rate constants for the uptake of unbound (free) T3 by the perfused rat liver, for the hepatic uptake of T3 from serum, and for the spontaneous dissociation of T3 from its plasma binding proteins. Quantitative autoradiography of liver lobules after perfusion with [125I]T3 in protein-free buffer indicated a high apparent rate constant for removal of T3 from the sinusoids; its minimum estimate was 2.4 +/- 0.2 sec-1. The single pass extraction of T3 in human serum by the perfused rat liver was 31.6 +/- 4.5% at the supraphysiological flow rate of 3 ml/min/g liver (sinusoidal transit time, approximately 3 sec). Sixty percent of the T3 in this serum dissociated spontaneously from its binding proteins in 3 sec, as determined by a rapid filtration assay. Based on these data, we conclude that the pool of free T3 in plasma turns over very rapidly in vivo and probably accounts for the entire hepatic uptake of T3 from plasma. Using additional data on the rate constant for cellular metabolism of T3 obtained from values reported in the literature, a previously published general mathematical model of ligand transport was applied to all of these data, yielding the following conclusions for the physiological state. 1) Metabolism, not uptake, is rate limiting to removal of T3 from plasma by the liver. 2) Intracellular T3 is in virtual equilibrium with the free T3 pool in plasma. 3) Intracellular T3 concentrations reflect the concentration of free T3 in plasma, as predicted by the free hormone hypothesis. It is shown mathematically that these conclusions are independent of whether a gradient exists between extra- and intracellular T3 concentrations, and that they would still hold even if the tissue uptake of T3 occurred by a mechanism that acted directly on the plasma protein-bound pool of T3.

Effects of dexamethasone on kinetics and distribution of triiodothyronine in the rat.

Studies were designed to examine the effects of the glucocorticoid dexamethasone (Dex) on the distribution and turnover of T3 separately from its effects on the pituitary and thyroid. Male Sprague-Dawley rats (200-250 g) were surgically thyroidectomized and given a replacement dose of T4 (1.6 micrograms/day X 100 g BW) throughout the experiment via a sc implanted osmotic minipump. Six or 7 days after starting the T4 infusion, each animal was given [125I]T3 by constant infusion (via a second minipump) for 5 or 6 days and, during the final 5 days, either Dex (0.15 mg/day X 100 g) or saline in a third minipump. Methanol extracts of serum and tissues removed at the end of the infusion were analyzed for [125I]T3 concentration by high performance liquid chromatography. The MCR, computed from the infusion rate of tracer and the serum concentration of [125I]T3 at the end of the infusion, averaged 25.7 +/- 1.3 (+/-SE) ml/h X 100 g in the controls and 15.1 +/- 2.6 in the Dex-treated rats. Serum T3 (RIA) concentrations were similar in the two groups. The plasma T3 production rate was decreased from 9.51 +/- 1.14 ng/h X 100 g in controls to 5.13 +/- 1.16 in the Dex-treated animals. The fraction of administered T4 converted to T3 was reduced from 0.21 to 0.11 by Dex treatment. Tissue to serum (T/S) [125I]T3 concentration ratios were significantly decreased by Dex to approximately 50% of the control value in each of the tissues sampled (liver, kidney, and skeletal muscle). The reduction in the T/S ratio could not be attributed to an increase in the net serum binding of T3; in fact, serum hormone binding was diminished by Dex treatment. The distribution data indicate that net tissue binding of T3 in these organs is reduced to an even greater extent than is serum binding of T3. The glucocorticoid-induced fall in T3 MCR could be accounted for by the decrease in T/S ratios, the latter being a measure of T3 distribution volume in the tissues studied. The rate of T4 5'-deiodination in vitro was diminished in homogenates of livers from Dex-treated animals when the incubation was performed with and without added thiol as cofactor, indicating that the hepatic level of active T4 5'-deiodinase is reduced by Dex. Thus, Dex causes multiple alterations in T3 metabolism. Total body T3 production from T4 in extrathyroid sites, and in the liver in particular, is reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbohydrate feeding increases total body and specific tissue 3,5,3'-triiodothyronine neogenesis in the rat.

The glucose-fed rat, in contrast to the chow-fed animal, has a higher serum total T3 concentration and an increase in the hepatic content of T4 5'-deiodinase (type I) activity. The mechanism and significance of these glucose-induced changes in T3 metabolism are elucidated in this study. To focus on extrathyroidal thyroid hormone metabolism the kinetic parameters were determined in thyroidectomized T4-replaced rats (1.25 micrograms T4/100 g BW.day). Kinetics of T4 and T3 were studied separately by infusing labeled hormone to equilibrium. Glucose feeding for 72 h (G) significantly increased both the total and free serum T3 concentrations compared to the respective means in the chow-fed control group (P). The glucose-induced changes in serum T3 reflect the approximate doubling of T3 production to 14.7 +/- 0.6 ng/h.100 g in G rats compared to 7.6 +/- 0.7 ng/h.100 g in P rats. The higher T3 production rate in the G group is due to a significant increase in the fractional total body T4 to T3 conversion (0.33 +/- 0.02) compared to that in the P group (0.19 +/- 0.02). The tissue (liver, kidney, brain, and brown adipose tissue) concentration of T4 (nanograms per g wet wt) was significantly increased in the G group. The increase ranged from 54% in liver to 80% in kidney, brain, and brown adipose tissue. The tissue concentration of T3 (nanograms per g wet wt) was even more dramatically increased by glucose feeding than was T4. The glucose-induced increment in organ T3 ranged from 2.5-fold (kidney, muscle, and brain) to 5-fold (liver and white adipose tissue) to 12-fold (brown adipose tissue). These data indicate that the increase in serum total and free T3 concentrations associated with glucose feeding reflects augmented total body T3 production from T4. The effect of the enhanced T3 neogenesis was generalized, as the T3 content was increased in each organ studied. Thus, glucose feeding has unique effects on T3 metabolism.

Thyroid hormone-binding proteins in plasma facilitate uniform distribution of thyroxine within tissues: a perfused rat liver study.

We used autoradiography to test the hypothesis that a major function of thyroid hormone-binding proteins in plasma is to ensure uniform distribution of thyroid hormones among cells of a given tissue. The distribution of [125I]T4 within rat hepatic lobules was determined after its single pass perfusion through the portal vein in solutions containing or lacking thyroid hormone-binding proteins. These proteins included thyroid hormone-binding globulin, thyroid hormone-binding prealbumin, and albumin. In the absence of these proteins, virtually all of the perfused T4 was taken up by the periportal cells, and subsequent perfusion with protein-free solution did not cause redistribution of this T4. In the presence of these proteins, in contrast, the perfused T4 was taken up uniformly by all cells within the lobule. Albumin alone was sufficient to ensure uniform cellular uptake of T4. However, variation of oleic acid concentrations within the physiological range markedly influenced the concentration of free T4 in a solution of 4% human serum albumin, but not in human serum. These results indicate that uniform distribution of T4 within tissues requires circulating thyroid hormone-binding proteins, and that the specific binding proteins, thyroid hormone-binding globulin and thyroid hormone-binding prealbumin, are required to ensure nonfluctuating circulating concentrations of free T4 in vivo. Other hormone-binding proteins in plasma and some transport proteins may function similarly.

Carbohydrate reactivation of thyroxine 5'-deiodinase (type II) in cultured mouse neuroblastoma cells is dependent upon new protein synthesis.

The T3 concentration in brain predominantly reflects local production from T4 rather than T3 uptake from the circulating pool. We recently demonstrated that rat brain T3 content is increased by glucose feeding compared to chow feeding. One possible mechanism for this effect is an increase in brain T4 5'-deiodinase (5'-D) activity. Our recent preliminary studies of neuroblastoma (NB) cells demonstrate that renewal of RPMI-1640 medium stimulates T4 5'-D type II (NB T4 5'-D II) activity in these cells. The present studies were performed to determine the mechanism of this response. Studies were performed on NB cells supported in thyroid hormone-depleted (deficient) medium. This approach increased NB T4 5'-DII activity 4-fold compared to that in thyroid hormone-replete medium. Medium renewal further stimulated enzyme activity (7- to 9-fold; maximum at 6 h) in each group. The difference between the hypothyroid group and control was sustained over a 24-h period. Subsequent studies demonstrated that glucose (11 mM) was the specific medium ingredient mediating the medium renewal response. A progressive increase in NB T4 5'-DII activity was noted over 8 h during RPMI-1640 salt plus glucose (11 mM) incubation. This was equivalent to the effect of complete medium containing glucose (11 mM). Coincubation with insulin (10(-7)-10(-9) M) did not modify the enzyme response to glucose. In addition, fructose (10 mM) had a similar effect on enzyme activity. Glycerol and essential and nonessential amino acids also modestly increased NB T4 5'-DII activity compared to that in the control group (P less than 0.01). Actinomycin-D (1 microM), cycloheximide (100 microM), and puromycin (100 microM) significantly (P less than 0.001) decreased the glucose effect on T4 5'-DII by 5-, 9-, and 17-fold, respectively, after 6 h of incubation. In addition, puromycin (10-200 microM) inhibited both NB T4 5'-DII activity and [3H]amino acid incorporation during incubation in glucose. There was a significant correlation between these parameters (r = 0.8; P less than 0.001). The enzyme activity decay curves in the glucose-activated and control groups subsequent to puromycin (100 microM) addition at 8 h were parallel. The fractional turnover rate was 13%/h in the controls and 11%/h in the glucose groups. The calculated enzyme production rate was significantly higher (P less than 0.005) in the glucose group compared to that in the control group (17.4 vs. 6.8 fmol/mg protein.h).(ABSTRACT TRUNCATED AT 400 WORDS)

Thyroid hormone export in rat FRTL-5 thyroid cells and mouse NIH-3T3 cells is carrier-mediated, verapamil-sensitive, and stereospecific.

Export of L-T3 out of the cell is one factor governing the cellular T3 content and response. We previously observed in liver-derived cells that T3 export was inhibited by verapamil, suggesting that it is due to either ATP-binding cassette/multidrug resistance (MDR1/mdr1b) or multidrug resistance-related (MRP1/mrp1) proteins. To test this hypothesis we measured T3 export in FRTL-5, NIH-3T3, and rat hepatoma (HTC) cells that varied in expression of these proteins. FRTL-5 and NIH-3T3 cells were found to contain a T3 efflux mechanism that is verapamil inhibitable, saturable, and stereospecific. By contrast, T3 efflux in HTC cells was slow and unaffected by verapamil. Neither FRTL-5 nor NIH-3T3 cells express mdrlb, but all three cell types express mrpl, as assessed by immunoblotting. Overexpression of MDR1 in NIH-3T3 cells did not enhance verapamil-inhibitable T3 efflux. Photoaffinity labeling of FRTL-5 and NIH-3T3 cells with [125I]L-T3 revealed a labeled 90- to 100-kDa protein that was not present in HTC cells. Verapamil and excess nonradioactive L-T3, but not D-T3, inhibited labeling of this protein. The lack of correlation between T3 efflux and MDR1 and mrpl expression and the finding of a photoaffinity-labeled putative transport protein smaller than MDR1 or mrp1 protein (approximately 170 kDa) suggest that a novel protein is involved in the transport of T3 out of cells.

Compensatory thyroid hypertrophy after hemithyroidectomy in rats.

Thyroid enlargement occurs in association with a variety of circumstances characterized by an impaired capacity of the gland to secrete adequate amounts of hormone. To elucidate the factors responsible for such compensatory thyroid growth, particularly the role of TSH, we have observed the response of the serum TSH, T3 and T4 concentrations following hemithyroidectomy in the rat, and have attempted to correlate changes in these functions with changes in the weight and histology of the thyroid remnant. Hemithyroidectomy was performed in male Sprague-Dawley rats weighing 150 to 370 g, sham-operated animals serving as controls. As compared to findings in sham-operated animals, serum T4 concentrations declined promptly after hemithyroidectomy. In Experiment I serum T4 concentrations remained low for about 10 days and then returned to initial values. In Experiment II serum T4 concentrations remained lower than initial T4 values or values found in sham-operated animals until 34 days after hemithyroidectomy. Serum T3 concentrations were not significantly altered after hemithyroidectomy in either group but tended to be lower in the hemithyroidectomized animals. Serum TSH concentrations increased within 3 days after hemithyroidectomy and, for as long as 21 weeks, remained at values higher than those present preoperatively or those seen in sham-operated animals. Thyroid lobe weight increased following removal of the contralateral lobe and this increase was also sustained throughout the duration of the experiments. Biochemical and histological observations indicated that enlargement of the residual lobe was due to hypertrophy rather than hyperplasia.

Uptake of cortisol by the perfused rat liver: validity of the free hormone hypothesis applied to cortisol.

The mechanism by which cortisol in plasma enters hepatic cells was investigated using the isolated perfused rat liver. To determine whether hepatic uptake of cortisol from serum can be accounted for entirely by the pool of unbound (free) cortisol, we compared observed uptake rates with the equilibrium-free fraction of cortisol in serum and the rates of dissociation of cortisol from its serum binding proteins (determined using a rapid filtration assay based on transfer of [3H] cortisol to dextran-coated charcoal). More than 95% of the cortisol in both human and rat serum dissociated spontaneously from its binding proteins within 5 sec at 37 C. The fractional unidirectional hepatic uptakes of cortisol from pooled human serum and pooled rat serum were 59.4 +/- 5.4% and 59.5 +/- 1.0% (mean +/- SE), respectively, at the physiological flow rate of 1 ml/min.g liver. The corresponding free cortisol fractions in these sera were 4.53 +/- 0.15% and 8.16 +/- 0.23%, respectively. The fractional unidirectional hepatic uptake of cortisol from protein-free buffer averaged 99.9% (n = 5) at a flow rate of 3 ml/min.g liver. By calculating the appropriate rate constants and applying the Kety-Renkin-Crone equation to the above data, it can be shown that all of the cortisol taken up from serum by the perfused rat liver can be accounted for by the pool of free cortisol, which turns over very rapidly. The physiological significance of this finding is discussed in terms of a general mathematical model of hormone transport that delineates the conditions under which the free hormone hypothesis is and is not valid.

Red blood cell thyroxine in nonthyroid illness and in heparin-treated patients.

Red blood cell T4 concentrations (RBC T4) were measured in 15 normal subjects, 13 patients with hypo- or hyperthyroidism, and 10 patients with elevated or decreased serum thyroid hormone binding. In each case, RBC T4 was compared with the serum concentration of free T4 measured by equilibrium dialysis ( FT4D ). RBC T4 correlated significantly with FT4D in these subjects (r = 0.90; P less than 0.001). The normal range for RBC T4 was 0.27-0.83 ng/ml. RBC T4 was below the normal range in all 8 patients with hypothyroidism and above the normal range in all 5 patients with hyperthyroidism. It was within the normal range in all 4 subjects with absent or low T4-binding globulin (TBG) and in 5 of the 6 subjects with elevated TBG or familial dysalbuminemic hyperthyroxinemia. The sixth subject (increased TBG) had elevated RBC T4 and FT4D . RBC T4 was similarly measured in 10 patients with severe nonthyroid illness (NTI), 5 of whom had decreased serum concentrations of total T4. RBC T4 was normal in 8 of these patients, elevated in 1, and decreased in 1; in comparison, FT4D was normal in 4, elevated in 5, and decreased in 1. Eight patients receiving continuous iv infusions of heparin were also studied because of previously described similarities in the in vitro thyroid tests of heparin-treated and euthyroid sick patients. FT4D was elevated in 7 of the heparin-treated patients, whereas RBC T4 was elevated in only 2. Furthermore, for any given value of FT4D , RBC T4 was lower in heparin-treated patients than in normal subjects, indicating the presence of an inhibitor of cellular T4 binding in these patients. This putative inhibitor, demonstrated by an elevated FT4D to RBC T4 ratio, was present in 6 of the 8 heparin-treated patients and in 5 of the 10 patients with NTI. The findings of this study support the hypothesis that an inhibitor of cellular T4 binding is present in the serum of some patients with NTI and in most heparin-treated individuals.

Thyroxine distribution and metabolism in familial dysalbuminemic hyperthyroxinemia.

We studied two families with familial dysalbuminemic hyperthyroxinemia (FDH), a recently described entity characterized by marked elevation of serum T4 due to increased binding of T4 to albumin. The seven affected subjects had elevated serum total T4 levels (range, 15.3-25.2 micrograms/dl; normal, 4.5-11.0 micrograms/dl), but normal serum free T4 levels, as measured by equilibrium dialysis. Their serum T3 levels ranged from 1.40-2.46 ng/ml (normal, 0.9-2.0 ng/ml). The proportion of T4 associated with serum albumin was increased approximately 4-fold in the affected subjects, as shown both by reverse flow paper electrophoresis and immunoprecipitation of albumin-bound T4 with antihuman serum albumin. In vivo T4 kinetic studies were performed in the two index subjects to assess the effects of the increased binding of T4 to albumin on the in vivo transport, distribution, and disposal of T4. Compared to values in normal subjects, the MCR of T4 was decreased by about 50%, and its total body (extrathyroidal) pool size was increased by approximately 50%; the T4 production rate was normal. The extracellular T4 pool size was increased by about 100% in the FDH subjects, but the rapidly exchangeable intracellular T4 pool size was normal. The unidirectional T4 clearance rate from plasma into the rapidly exchangeable cellular compartment was reduced by approximately 50%, but the absolute rate of T4 flux from plasma into the cellular compartment was normal. Thus, the in vivo kinetic data indicate that the increased plasma binding of T4 in FDH alters the distribution of T4 in favor of the extracellular compartment, retards the fractional rate of transfer of T4 into cells, and slows the metabolic clearance of T4. However, the absolute rate of T4 flux into the rapidly exchangeable cellular compartment, the intracellular T4 pool size, and the T4 disposal rate are all normal in FDH, consistent with the normal serum concentrations of free T4 and the eumetabolic state of these individuals.

The acute effects of human growth hormone administration on thyroid function in normal men.

GH replacement therapy may lead to alterations in serum TSH and/or thyroid hormone values in GH-deficient patients, but there is no consensus on the explanation for these changes. We examined the effect of GH administration (0.125 mg, sc, daily for 4 days) on thyroid function in 20 normal men. Serum T4 levels decreased by 8%, and serum free T4 index values decreased by 5%. In contrast, serum T3 levels increased by 21%; serum rT3 did not change. These changes were accompanied by a 54% decrease in the mean serum TSH level. While it is not possible to draw conclusions about hormone production and disposal rates from changes in serum levels, these data are most consistent with enhanced extrathyroidal (including intrapituitary) conversion of T4 to T3 and a compensatory decrease in TSH secretion.

Effect of free fatty acids on the concentration of free thyroxine in human serum: the role of albumin.

The concentration of FFA in normal human plasma in vivo generally ranges between 0.2 and 0.7 meq/liter; slightly higher concentrations have occasionally been reported in patients who are seriously ill. To determine whether such FFA concentrations may increase the concentration of free T4 in serum, we added increasing amounts of oleic acid to pooled normal human serum (with known FFA content) and measured free T4 by equilibrium dialysis. Total FFA up to 3 meq/liter in normal serum, representing an FFA to albumin molar ratio of about 5:1, had little or no effect on the free T4 concentration, while higher FFA concentrations progressively increased free T4. This same molar ratio of FFA to albumin had to be exceeded to cause a significant increase in the free T4 concentration in diluted serum and in serum from patients with nonthyroid illness. Serum from which more than 95% of the albumin had been removed by chromatography with Affi-Gel blue was much more sensitive to the effects of FFA on free T4. This enhanced sensitivity was reversed by readdition of albumin to the serum, and the addition of albumin to normal serum resulted in diminished effects of FFA on free T4. These results indicate the following: physiological concentrations of FFA do not significantly increase the free T4 concentration in normal human serum; when FFA reach supraphysiological concentrations in serum (in vitro) and the higher affinity FFA-binding sites on albumin become saturated (apparently at an FFA to albumin molar ratio of approximately 5:1), the excess FFA interact with other serum proteins, including thyroid hormone-binding globulin, and thereby increase the free T4 concentration; the concentration of albumin (or other FFA binders) must be considered when evaluating the observed effects of FFA. To explore the relevance of these findings to the hypothesis that FFA may inhibit the binding of T4 to plasma proteins in patients with nonthyroid illness, we measured plasma FFA concentrations in 11 severely ill patients hospitalized in the intensive care unit. We found a mean plasma FFA concentration of 0.45 +/- 0.11 (+/- SEM) mEq/liter and a mean serum albumin concentration of 2.39 +/- 0.29 g/dl in these patients. Their mean plasma FFA to albumin molar ratio was 1.53 +/- 0.41. Since the FFA to albumin molar ratio must exceed about approximately 5:1 before a significant increase in the serum free T4 concentration occurs, these results suggest that FFA do not commonly influence the circulating free T4 concentration in vivo, even in severely ill patients.

Mechanism of the heparin-induced increase in the concentration of free thyroxine in plasma.

The iv administration of heparin causes an increase in the plasma free T4 concentration, as determined by equilibrium dialysis. The mechanism and physiological consequences of this action of heparin are unknown. To explore the possibility that the heparin-induced increase in plasma free T4 is an in vitro artifact due to generation of FFA during equilibrium dialysis, we studied plasma samples from 10 subjects treated with iv heparin. In plasma from 4 of these subjects, free T4 concentrations measured by equilibrium dialysis did not increase above baseline values after heparin administration. In incubations performed in parallel with the equilibrium dialysis measurements, FFA concentrations in these plasma samples were found to increase, but in no subject did they exceed 2.5 meq/L after incubation. In contrast, in plasma from the other 6 subjects treated with heparin, free T4 concentrations rose markedly (by 130-520%) above baseline values after heparin administration. In all of these postheparin plasma samples, FFA concentrations were less than 2.8 meq/L before incubation, but rose during incubation by 80-270% to more than 3.8 meq/L. Treatment of these plasma samples with protamine to inhibit lipoprotein lipase and with specific antiserum to inhibit hepatic triglyceride lipase before equilibrium dialysis or incubation prevented, in parallel, the heparin-induced increases in FFA and free T4 concentrations. From these findings we conclude that the heparin-induced increase in free T4 is usually an in vitro artifact, and that most subjects receiving heparin have a normal plasma free T4 concentration in vivo. We also conclude that this in vitro artifact may account for many of the findings that led to the postulate of an inhibitor of T4 binding to plasma and intracellular proteins in heparin-treated patients and perhaps in patients with nonthyroid illness as well.

Extrathyroidal conversion of thyroxine to 3,3',5'-triiodothyronine (reverse-T3) and to 3,5,3'-triiodothyronine (T3) in humans.

In order to estimate the relative magnitude of the two alternative pathways of monodeiodination of thyroxine (T4) in adult humans, the metabolic clearance rates (MCR) and production rates (PR) of 3,3',5'-triiodothyronine (reverse-T3,rT3) and of 3,5,3'-triiodothyronine (T3) were determined in six euthyroid control subjects (C) and in five hypothyroid patients (H) receiving L-T4 as replacement therapy (0.15-0.3 mg/day). MCR was computed by a non-compartmental method of analysis from the plasma disappearance of 125I rT3 and 131I T3 during 72 h following simultaneous injection of tracers. PR was calculated from MCR and the serum concentration of rT3 and T3, respectively, determined by radioimmunoassay. In the H subjects, rT3 MCR averaged 97.1 +/- 12.8 (SD) 1/day and rT3 PR, 34.3 +/- 12.8 microng/day; T3 MCR was 28.7 +/- 6.1 1/day and T3 PR, 20.3 +/- 6.6 microng/day (all corrected to 70 kg body weight). These results were not significantly different from those in the control group; rT3 MCR 104 +/- 24 1/day, rT3 PR 33.0 +/- 9.2 microng/day; T3 MCR 24.0 +/- 5.9, T3 PR 24.2 +/- 4.1. The proportionof total triiodothyronine (rT3 averaged 62% in H patients and was similar (57%) in the C group. The results obtained in the H subjects indicate that the production of rT3 is a major route of T4 metabolism, equal to or exceeding that of T3. From the close agreement between the mean values for rT3 PR in the C and H groups it is concluded that most, if not all of the rT3 produced in normal humans is derived by extrathyroidal conversion from T4.